The goal of this project is to address several unresolved questions regarding the role of the endocannabinoid system in pain, reward and drug dependence. There is strong evidence that the two major endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) exert considerable influence in these pathological states, yet it is highly unlikely that they are simply playing redundant roles. Therefore, one of the objectives of this project is to delineate their roles in these pathological conditions. The fact that the synthetic and metabolic pathways for endocannabinoids are not completely understood creates a challenge in systematically manipulating their levels under in vivo conditions. Therefore, a major goal is to develop potent and selective enzyme inhibitors that can be used to manipulate endocannabinoids in vivo. Synthetic and metabolic enzyme inhibitors for AEA and 2-AG will be prepared by Dr. Razdan (Project 2). Those identified by Dr. Cravatt (Project 3) as enzyme selective will be evaluated by us in behavioral battery of tests for cannabinoid activity. The second approach is to establish the phenotypes of mice deficient in endocannabinoid synthetic and metabolic enzymes generated by Dr. Cravatt. At the same time, we are well aware that several classes of lipids structurally related to AEA and 2-AG impact the endocannabinoid system by influencing synthetic and metabolic pathways, acting directly on cannabinoid receptors, or acting at the newly discovered allosteric site on the CB{1} receptor site. To address these questions, we will conduct in vitro and in vivo evaluation of synthetic allosteric ligands for CB{1} receptors [prepared by Drs. Razdan and Mechoulam (Project 4)], and putative endocannabinoids provided by Dr. Mechoulam. To complement Dr. Mechoulam's efforts to identify new endocannabinoids, we will establish lipid profiles in selected brain regions of mice under different experimental conditions in an effort to identify lipids that may be endocannabinoids or relevant lipid mediators. We will use existing as well as these new discoveries to further explore the involvement of the endocannabinoid system in pain, reward and dependence. Emphasis will be placed on inflammatory and neuropathic pain models. We will determine the extent to which cytokines and chemokines are involved in endocannabinoid anti-inflammatory effects. The receptor mechanisms of action and underlying neural substrates will be investigated using genetic and pharmacological approaches. The same comprehensive approach will be employed in drug discrimination and feeding behavior, to establish THC-like profiles of agents that manipulate the endocannabinoid system and to establish phenotypic behavior of genetically modified mice. Collectively, these studies will identify endogenous substances that may act either directly or indirectly on the endogenous cannabinoid system, and elucidate the role that AEA and 2-AG play in pain, reward, and drug dependence.